Advances in imaging biological cells using optical tomography have been developed by Nelson as disclosed, for example, in U.S. Pat. No. 6,522,775, issued Feb. 18, 2003, and entitled “Apparatus and method for imaging small objects in a flow stream using optical tomography,” the full disclosure of which is incorporated by reference. Further developments in the field are taught in Fauver et al., U.S. Pat. No. 7,738,945 issued Jun. 15, 2010, entitled “Method and apparatus of shadowgram formation for optical tomography,” (Fauver '945) and Fauver et al., U.S. Pat. No. 7,907,765 issued Mar. 15, 2011, entitled “Focal plane tracking for optical microtomography,” the full disclosures of which are also incorporated by reference.
Processing in an optical tomography system begins with specimen preparation. Typically, specimens taken from a patient are received from a hospital or clinic and processed to remove non-diagnostic elements, fixed and then stained. Stained specimens are then mixed with an optical gel, inserted into a microcapillary tube and images of objects, such as cells, in the specimen are produced using an optical tomography system. The resultant images comprise a set of extended depth of field images from differing perspectives called “pseudoprojection images.” The set of pseudoprojection images can be reconstructed using backprojection and filtering techniques to yield a 3D reconstruction of a cell of interest. The ability to have isometric or roughly equal resolution in all three dimensions is an advantage in 3D tomographic cell imaging, especially for quantitative image analysis.
The 3D reconstruction then remains available for analysis in order to enable the quantification and the determination of the location of structures, molecules or molecular probes of interest. An object such as a biological cell may be labeled with at least one stain or tagged molecular probe, and the measured amount and location of this biomarker may yield important information about the disease state of the cell, including, but not limited to, various cancers such as lung, breast, prostate, cervical, stomach and pancreatic cancers.
The present disclosure allows an extension of optical projection tomography to live cell imaging and is expected to advance cell analysis, drug development, personalized therapy, and related fields. Until now, live cell microscopy has traditionally been done by non-labeling 2D imaging techniques such as phase contrast, DIC, and polarization contrast microscopy.
Native absorbance and fluorescence imaging using deep ultraviolet (DUV) at 250 nm to 290 nm wavelengths has been technically challenging and causes phototoxicity in irradiated cells. More recently, vital stains have been used that typically emit fluorescence signals for 3D live cell imaging, because commercial microscopes (of confocal, deconvolution, and multiphoton excitation varieties) rely on fluorescence for building up multiple planar slices for generating 3D images. However, in these cases, the 3D image resulting from a stack of 2D images has about four times less axial resolution as the lateral resolution within each slice, thereby making quantitative analysis imprecise. The ability to have isometric or roughly equal resolution in all three dimensions is a significant advantage in 3D tomographic cell imaging, especially for quantitative image analysis.
One advantage of using DUV illumination for live cells is that native DNA and protein absorb the light at 260 nm and 280 nm, respectively, without the use of any photochemical label that must permeate the cell membrane and sometimes the nuclear membrane of the cell, which is in a non-normal state. Furthermore, the label or stain is only an intermediary step toward the measurement of target protein or nucleotide (DNA) which adds a large degree of variability in this measurement. Elimination of such exogenous species would potentially improve the accuracy of a quantitative measure of protein or nucleotide (DNA), as well as reduce time, effort and complexity by eliminating steps in the sample preparation. Unfortunately, the use of DUV illumination has demonstrated phototoxicity in the past, due to the high dose of radiation required to stimulate a strong signal.
Recently, however, DUV imaging of live cultured human and mouse cells was demonstrated at 260 nm and 280 nm using DUV light-emitting diodes (LEDs) (See, for example, Zeskind, B J, et al., “P. Nucleic acid and protein mass mapping by live cell deep ultraviolet microscopy,” Nature Methods 4(7):567-569 (2007)).
The present disclosure describes a new, novel and surprisingly effective 3D imaging system that provides solutions to long felt needs in the field of DUV 3D imaging of cells, and more particularly, live cells.